Voltage clamp analysis of tetrodotoxin-sensitive and -insensitive sodium channels in rat muscle cells developing in vitro

Sodium currents in cultured rat muscle cells converted to myoballs by treatment with colchicine were recorded using a giga-ohm seal voltage clamp procedure in the whole cell configuration. The mean peak Na⁺ conductance of the myoballs was 90 pS/μm² of surface membrane. Half-maximal activation of Na⁺ currents was observed for test pulses to -31 mV and half-maximal inactivation was observed for prepulses to -74 mV. Titration of the inhibition of Na⁺ currents by tetrodotoxin (TTX) yielded a biphasic inhibition curve consistent with the presence of two classes of Na⁺ channels differing in affinity for TTX. The TTX-sensitive channels carried 28% of the Na⁺ current and had an apparent K(D) for TTX of 13 nM at 20°C. The TTX-insensitive Na⁺ channels had an apparent K(D) for TTX of 3.2 μM. Inhibition of TTX-insensitive Na⁺ channels by TTX was enhanced by repetitive stimulation of the myoballs at 2 Hz, whereas the inhibition of TTX-sensitive Na⁺ channels by TTX was not frequency dependent. We conclude that rat muscle cells developing in vitro synthesize physiologically functional, TTX-sensitive Na⁺ channels in the absence of innervation. These channels, which are characteristic of adult skeletal muscle, function in parallel with TTX-insensitive Na⁺ channels that are present in embryonic muscle.